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. 1998 Oct;18(10):6110-20.
doi: 10.1128/MCB.18.10.6110.

Functional characterization of the N terminus of Sir3p

M Gotta  1 , F Palladino, S M Gasser
Affiliations

Functional characterization of the N terminus of Sir3p

M Gotta et al. Mol Cell Biol. 1998 Oct.

VSports在线直播 - Abstract

Silent information regulator 3 is an essential component of the Saccharomyces cerevisiae silencing complex that functions at telomeres and the silent mating-type loci, HMR and HML. We show that expression of the N- and C-terminal-encoding halves of SIR3 in trans partially complements the mating defect of the sir3 null allele, suggesting that the two domains have distinct functions. We present here a functional characterization of these domains. The N-terminal domain (Sir3N) increases both the frequency and extent of telomere-proximal silencing when expressed ectopically in SIR+ yeast strains, although we are unable to detect interaction between this domain and any known components of the silencing machinery. In contrast to its effect at telomeres, Sir3N overexpression derepresses transcription of reporter genes inserted in the ribosomal DNA (rDNA) array VSports手机版. Immunolocalization of Sir3N-GFP and Sir2p suggests that Sir3N directly antagonizes nucleolar Sir2p, releasing an rDNA-bound population of Sir2p so that it can enhance repression at telomeres. Overexpression of the C-terminal domain of either Sir3p or Sir4p has a dominant-negative effect on telomeric silencing. In strains overexpressing the C-terminal domain of Sir4p, elevated expression of either full-length Sir3p or Sir3N restores repression and the punctate pattern of Sir3p and Rap1p immunostaining. The similarity of Sir3N and Sir3p overexpression phenotypes suggests that Sir3N acts as an allosteric effector of Sir3p, either enhancing its interactions with other silencing components or liberating the full-length protein from nonfunctional complexes. .

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Figures

FIG. 1
FIG. 1
Overexpression of Sir3N restores sectoring in Sir4C-overexpressing cells. The strain AJL275-2AVIIL, which carries ADE2 adjacent to the VIIL telomere, was transformed with pADH-SIR4C (also called pFP340) and pAAH5 (a); pADH-SIR4C and pADH-SIR3N (b); pADH-SIR4C and pADH-SIR3 (p2μ-ASIR3 [26]) (c); control vectors (pAAH5 and p2HG) (d); pADH-SIR3N and p2HG (e); and pADH-SIR3 (p2μ-ASIR3 [26]) and p2HG (f). In all cases two plasmids were present and isolated colonies were streaked onto medium lacking histidine and leucine to ensure maintenance of the plasmids. Adenine concentrations are limiting. The colonies were allowed to grow for 5 days at 30°C. Following incubation, the plates were stored at 4°C to enhance the pigmentation of the cells.
FIG. 2
FIG. 2
Functional domains of Sir3p. A schematic representation of full-length Sir3p and the functional domains revealed by genetic, two-hybrid, and biochemical studies is shown. Notes: 1, reference ; 2, two-hybrid data indicate that the Sir4p binding domain is 3′ of aa 494 (6), and unpublished pull-down data indicate that there is only one site of interaction, not two as previously suggested (12a, 43), 3, reference (as indicated by the shaded box, the domain necessary and sufficient for Rap1p interaction has been narrowed down to aa 455 to 481 of Sir3p, and the Sir3p homodimerization domain has been defined from aa 762 to the end of the protein [38a]); 4, reference ; 5, this study. The two mutations isolated as suppressors of histone H4 mutants are labeled SIR3R1 and SIR3R3 (18). See the text for more details.
FIG. 3
FIG. 3
Overexpression Sir3N counteracts the effect of overexpression of Sir3C at telomeres. The strain UCC3107, which carries ADE2 adjacent to the VR telomere, was transformed with plasmids as indicated as well as a second control plasmid. The control plasmids are the backbone vectors without SIR gene inserts, namely, pAAH5 and p423ADH (see Materials and Methods). Plasmids with SIR3 gene inserts are pADH-SIR3N and pADH-SIR3C (pMG17). In each case, two independent transformants were streaked onto medium lacking histidine and leucine to ensure maintenance of the plasmids and with limiting adenine concentrations. The cells were allowed to grow for 3 to 5 days at 30°C, and the plates were stored at 4°C to enhance the pigmentation of the cells.
FIG. 4
FIG. 4
Sir3N overexpression promotes TPE spreading. Strains UCC518, UCC520, and UCC522 were transformed with pADH (pAAH5), pADH-SIR3N, and pADH-SIR3 (p2μ-ASIR3 [26]). Colonies were grown for 3 to 5 days at 30°C on medium lacking leucine, to ensure maintenance of the plasmids, and resuspended in H2O, and 10-fold serial dilutions were plated onto medium lacking leucine (−leu) and medium lacking leucine and containing 1 mg of 5-FOA/liter (5-FOA −leu), as described in Materials and Methods. Two independent transformants for each case are shown. On the right is a schematic representation of the URA3-marked telomere of strain UCC518 (top; URA3 at 2 kb from the telomere), UCC520 (middle; URA3 at 4 kb), and UCC522 (bottom; URA3 at 6 kb). The frequency of resistance to 5-FOA was calculated from eight independent transformants, each scored in three independent assays. The means and standard deviations are given in the text.
FIG. 5
FIG. 5
Sir3N derepresses rDNA silencing. Strain JS231 carrying the URA3 gene inserted at the rDNA was transformed with plasmids pADH (pAAH5), pADH-SIR3N, pADH-SIR3C, and pADH-SIR3 (p2μ-ASIR3). Colonies were grown for 3 to 5 days at 30°C on medium lacking leucine, to ensure maintenance of the plasmids, and resuspended in H2O, and 10-fold serial dilutions were plated onto medium lacking leucine (−leu) and medium lacking leucine and uracil (−leu −ura), as described in Materials and Methods. Eight independent transformants for each plasmid were tested, of which two are shown.
FIG. 6
FIG. 6
(A) SIR3N-GFP localizes to the nucleolus. The haploid strain UCC3107 was transformed with either a 2μ-based plasmid (a, b, and c) or a centromeric plasmid (d and f) expressing Sir3N fused to the green fluorescent protein under the control of the ADH promoter (pSIR3N-GFP2 and pSIR3N-GFP1, respectively). The direct fluorescence of Sir3N-GFP (green) (a), anti-Nop1p staining on the same cells visualized with a Cy3-conjugated secondary antibody (red) (b), and the merge of the two stainings (c) are shown. The blue area represents the nucleus. Colocalization of Sir3N-GFP and Nop1p is white. Cells transformed with pSIR3N-GFP1 and stained with anti-GFP antibodies (the kind gift of K. E. Sawin, Imperial Cancer Research Fund, London, England) visualized by a DTAF-conjugated secondary antibody (green) (d); the DNA staining of the same cells with POPO-3, which preferentially stains the nucleolar domain (red) (e); and anti-Sir2p immunofluorescence on a fixed wild-type diploid strain (GA229) that had been washed in 1% Triton–0.02% sodium dodecyl sulfate as described previously (10) (f) are also shown. Sir2p staining is visualized by a DTAF-conjugated secondary antibody (green), and the DNA is counterstained with ethidium bromide. Immunofluorescence assays were performed with affinity-purified antibodies as described in Materials and Methods. The arrows indicate an apparent looped body, while the arrowhead indicates the same loop extended. (B) SIR2 but not SIR4 is necessary for the enrichment of Sir3N-GFP in the nucleolus. The haploid strains UCC3107 (SIR+) (a and a′), UCC3203 (sir2::HIS3) (b and b′), and UCC3207 (sir4::HIS3) (c and c′) were transformed with plasmid pSIR3N-GFP1. The phase-contrast image (a to c) and the direct fluorescence (a′, b′, and c′) of Sir3N-GFP are shown. Results identical to those shown in c and c′ were obtained for a sir3::HIS3 strain.
FIG. 7
FIG. 7
Sir2p is enriched at telomeric foci upon overexpression of Sir3N. The wild-type haploid strain UCC3107, transformed with plasmids pADH (pAAH5) and pADH-SIR3N, was stained with anti-Sir2p, detected by a DTAF-conjugated secondary antibody (green). The DNA was counterstained with POPO-3 (red).
FIG. 8
FIG. 8
Overexpression of full-length Sir3p restores silencing in a strain that overexpress full-length Sir4p. The strain UCC3107, which carries ADE2 adjacent to the VR telomere, was transformed with plasmids as indicated as well as a second control plasmid. The control plasmids are the backbone vector without SIR gene inserts, namely, pAAH5 and p2HG (see Materials and Methods). The plasmids with SIR gene inserts are pADH-SIR4 and pGPD-SIR3. Colonies (two independent transformants in each case) were streaked onto medium lacking histidine and leucine to ensure maintenance of the plasmids and allowed to grow for 3 to 5 days at 30°C. Following incubation, the plates were stored at 4°C for 1 week to enhance the pigmentation of the cells.
FIG. 9
FIG. 9
Overexpression of Sir3N restores the focal staining pattern of Sir3p. Yeast cells were stained with anti-Sir3p antibodies detected by a DTAF-conjugated secondary antibody (white signal). All signals are within the yeast nuclei, as indicated in the insets, where the anti-Sir3p signals are superimposed on a DNA stain to reveal the nuclear shape (see Materials and Methods). Strain EG37 (26) transformed with the vectors pAAH5 and pRS316 (a), pC-ASir4 (26) and pAAH5 (b), and pC-ASir4 and p2μ-ASir3 (c) and strain AJL275-2AVIIL transformed with the vectors pAAH5 and p2HG (d), pADH-SIR4C (pFP340) and pAAH5 (e), and pADH-SIR4C (pFP340) and pADH-SIR3N (f) are shown.
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